Environmentally-friendly preparation method of composition containing anti-inflammatory active ingredients and use of composition

ABSTRACT

An environmentally-friendly preparation method of a composition containing anti-inflammatory ingredients and use of the composition are provided. The method includes: S1. weighing a specified volume of water and adjusting the pH, adding Sophora flavescens roots, and subjecting a resulting mixture to extraction at a high temperature; S2. adjusting pH of an obtained solution in step S1, adding Glycyrrhiza inflata roots, and heating a resulting mixture to boiling for extraction; S3. adding Scutellaria baicalensis roots to an obtained boiling solution from the extraction in step S2, and subjecting a resulting mixture to extraction, filtration, and pH adjustment; S4. adding activated carbon to an obtained filtrate in step S3, and subjecting a resulting mixture to incubation and filtration; S5. adding a clarifying agent to an obtained filtrate in step S4, and subjecting a resulting mixture to standing and filtration; and S6. subjecting an obtained filtrate in step S5 to membrane separation.

CROSS REFERENCE TO THE RELATED APPLICATIONS

This application is the national phase entry of InternationalApplication No. PCT/CN2020/120294, filed on Oct. 12, 2020, which isbased upon and claims priority to Chinese Patent Application No.201911045380.X, filed on Oct. 30, 2019, the entire contents of which areincorporated herein by reference.

TECHNICAL FIELD

The present invention relates to the technical field of biomedicine andcosmetics, and in particular to an environmentally-friendly preparationmethod of a composition containing anti-inflammatory active ingredientsand use of the composition.

BACKGROUND

Scutellaria baicalensis has long been used for diseases such as seasonalfebrile disease, pneumonia, abscesses, and furunculosis due to itseffects of clearing away pathogenic heat and dampness, purging intenseheat, and detoxification. Sophora flavescens has the functions ofclearing away pathogenic heat and dampness, killing insects, andpromoting urination, and is used for eczema, pruritus, and so on.Glycyrrhiza has the effects of clearing away heat and toxic materials,expelling phlegm and subdue coughing, spasm and pain relief, andreconciling various medicines. Glycyrrhizic acid shows antagonism tomany links such as the occurrence, development, and response ofinflammation. However, different extraction methods may result indifferent compositions of active ingredients, which may lead todifferent effects. Traditionally, the three medicines require to beco-decocted just before administration, which is troublesome,unfavourable for long-term storage, and inconvenient for use.

There have been a large number of reports on the extraction andpurification process for the main active ingredients in each of Sophoraflavescens, Scutellaria baicalensis, and Glycyrrhiza. The main methodsinclude water extraction, alcohol extraction, and the like. However, forlarge-scale production, ethanol is a Class A dangerous article and needsto be stored in a warehouse designed as a Class A fireproof building atan amount that should not be large. Therefore, in a production process,less or no ethanol should be used in consideration of environmentalprotection and economy. The water extraction method has low efficiencyand is prone to deterioration and bacterium growth in a high temperatureenvironment. In large-scale production, it is necessary to considershortening a process, improving efficiency, saving energy, andprotecting the environment.

There are no detailed reports in patents on the extraction of activeingredients simultaneously from any two of Sophora flavescens,Scutellaria baicalensis, and Glycyrrhiza, as well as few related reportsin literatures.

For example, Chinese patent CN2016109373139 “Traditional ChineseMedicine Extract Composition with Anti-skin Irritation Effect andPreparation Method Thereof” discloses a method for subjectingGlycyrrhiza, Sophora flavescens, Scutellaria baicalensis, Angelicasinensis, Ligusticum wallichii, Herba Schizonepetae, RadixSaposhnikoviae, and White Willow Bark to ethanol extraction, which isdifferent from an aqueous extraction method where medicinal materialsare fed in a specified order according to the properties of activesubstances in each medicinal material.

For example, in literature 1 ((Lu Bing, Yang Xuan, Wang Baohua, et al.Research on solubilization of glycyrrhizic acid on baicalin [J]. Journalof Beijing University of Traditional Chinese Medicine, 2014, 37 (9):620-624.), a method for subjecting Glycyrrhiza and Scutellariabaicalensis to heat reflux co-extraction with water to increase a yieldof baicalin, with a glycyrrhizic acid yield of 0.22 g/L and a baicalinyield of 164 mg/38 ml.

In literature 2 (Zhao Yang, Zhang Tao, Jia Hongmei, et al. Optimizationof Extraction Process for Medicine Pair of Sophoraflavescens-Glycyrrhiza and Analysis of Chemical Components therefrom[J]. Chinese Journal of Experimental Traditional Medical Formulae, 2017(03): 26-32.), a method for increasing the yields of Sophora flavescenstotal alkali and glycyrrhizic acid is provided, where, Glycyrrhiza andSophora flavescens are mixed at a ratio of 1:1 and subjected to refluxco-extraction with 60% ethanol.

In literature 3 (Liu Bin, Shi Renbing, Zhu Lijun, et al. Study on HPLCfingerprint of flavonoids in Sophora flavescens Decoction andCorrelation of HPLC fingerprint with Scutellaria baicalensis and Sophoraflavescens in Formula [J]. China Journal of Chinese Materia Medica,2007, 32 (16): 1631-1634.), a method for subjecting a mixture of Sophoraflavescens-Scutellaria baicalensis-Radix Rehmanniae (3:2:8) to refluxco-extraction with 70% ethanol and studying corresponding fingerprintsis provided, but no experiment is conducted to illustrate a relationshipbetween extracted biologically active ingredients and drug effects.

However, the above works of literature do not involve a method forsubjecting the three medicinal materials of Sophora flavescens,Scutellaria baicalensis, and Glycyrrhiza to co-extraction with anaqueous solution, which has a simplified process and reduced steps, hasa reduced contamination risk, and can achieve energy conservation andenvironmental protection. In addition, in the above works of literature,no specific solutions are proposed specifically for the stability ofsolvents, devices, solution products that require attention inlarge-scale production and for the efficacy changes caused by processchanges.

SUMMARY

In view of the shortcomings in the prior art, the present invention isintended to provide an environmentally-friendly preparation method of acomposition containing anti-inflammatory active ingredients.Specifically, in preparation, three medicinal materials of Sophoraflavescens roots, Scutellaria baicalensis roots, and Glycyrrhiza inflataroots are fed in a specified order for co-extracting biologically activeingredients, so as to achieve the following effects:

1) The present invention has a simplified process and no organicsolvents, results in no wastewater and a reduced contamination risk, andis energy-saving as well as environmentally-friendly.

2) Moreover, through the improvement of a process involved in thepresent invention, the application of a product in biomedicines, foodand health products, and cosmetics can be expanded, especially, theeffect on skin can be enhanced and expanded.

The objectives of the present invention are achieved by the followingtechnical solutions.

The present invention provides an environmentally-friendly preparationmethod of a composition containing anti-inflammatory active ingredients,including the following steps:

S1. weighing a specified volume of water and adjusting pH, addingSophora flavescens roots, and subjecting a resulting mixture toextraction at a high temperature;

S2. adjusting pH of an obtained solution from the extraction in step S1,adding Glycyrrhiza inflata roots, and heating a resulting mixture toboiling for extraction;

S3. adding Scutellaria baicalensis roots to an obtained boiling solutionfrom the extraction in step S2, and subjecting a resulting mixture toextraction, filtration, and pH adjustment;

S4. adding activated carbon to an obtained filtrate in step S3, andsubjecting a resulting mixture to incubation and filtration;

S5. adding a clarifying agent to an obtained filtrate in step S4, andsubjecting a resulting mixture to standing and filtration; and

S6. subjecting an obtained filtrate in step S5 to membrane separation toobtain the composition containing anti-inflammatory active ingredients.

Preferably, the Scutellaria baicalensis roots, Sophora flavescens roots,and Glycyrrhiza inflata roots may be added at a ratio of(21-40):(21-40):(21-40). Within the material ratio range, thecomposition containing anti-inflammatory active ingredients can beobtained.

In the present invention, Glycyrrhiza inflata roots are specificallyselected for compound extraction with Scutellaria baicalensis roots andSophora flavescens roots, which can achieve a superior anti-inflammatoryeffect and lead to a final product with high stability. However, ifother licorice roots such as Glycyrrhiza uralensis roots or Glycyrrhizaglabra roots are selected for compound extraction with Scutellariabaicalensis roots and Sophora flavescens roots, an obtained compositionexhibits an anti-inflammatory effect inferior to that of the presentinvention, and ingredients in the licorice roots will negatively affectthe color, appearance, and stability of a final product.

Preferably, in step S1, the water may be added at an amount 5 to 10times the total amount of medicinal materials; the pH may be 3.0 to 7.0;and the high-temperature extraction may be conducted at 60° C. to 80° C.for 30 min to 2 h.

Preferably, in step S2, the pH may be adjusted to 8 to 10 with 1% to 10%NaOH, and the boiling solution may be subjected to extraction for 30 minto 2 h. The slightly alkaline condition resulting from matrine isconducive to the dissolution of glycyrrhizic acid, and the surfaceactivity of glycyrrhizic acid is conducive to the dissolution ofmatrine. Moreover, with a slightly alkaline pH, matrine is not destroyedand glycyrrhizic acid is in an ionic state, which is more conducive tothe dissolution of glycyrrhizic acid and other active ingredients.

Preferably, in step S3, the extraction may be conducted for 30 min to 2h, and the pH may be adjusted to 5.5 to 7.5.

In this step, the Scutellaria baicalensis roots include specialbaicalinase, which tends to hydrolyze baicalin, causing the reduction ofbaicalin. Therefore, it is necessary to use boiling to inactivatebaicalinase, thus retaining the component of baicalin and enhancing theefficacy. In addition, baicalin is a flavonoid compound with multiplephenolic hydroxyl groups, and plant cell walls are easily ruptured at apH of 8 to 10, thus increasing the dissolution of flavonoids inScutellaria baicalensis roots. Under alkaline conditions, matrine canform hydrogen bonds with baicalin to enhance the solubility of baicalin.Moreover, glycyrrhizic acid can increase a surface tension of asolution, which improves the extraction efficiency of matrine andbaicalin.

In the present invention, it is found through preliminary tests:

1. In a traditional method for co-decocting the three medicinalmaterials, Scutellaria baicalensis roots, Sophora flavescens roots, andGlycyrrhiza inflata roots are fed at the same time, then waterextraction is conducted, pH of a resulting filtrate is adjusted to 5.5to 7.5, and then the filtrate is subjected to the treatments of steps S4to S6. Experimental results show that filtration is difficult in theprocess, causing difficulties in a production process.

2. In a case where the three materials are added in a changed order, forexample:

A. Water is added and pH is adjusted to 8 to 10 with 10% NaOH;Glycyrrhiza inflata roots are first added for extraction, then Sophoraflavescens roots are added for extraction, and finally Scutellariabaicalensis roots are added for extraction; a resulting solution isfiltered to obtain a filtrate and pH of the filtrate is adjusted to 5.5to 7.5; and then the filtrate is subjected to the treatments of steps S4to S6. Under slightly alkaline conditions, matrine is in a free stateand has insufficient water solubility, and the present invention adoptswater extraction from the perspective of environmental protection, so acontent of the active ingredient matrine in a water extraction solutionis reduced. Compared with the process of the present invention, anextraction yield of matrine is reduced by 32.3%.

Provided that after Glycyrrhiza inflata roots are subjected toextraction, pH of a resulting solution is first adjusted to acidity andthen Sophora flavescens roots are subjected to extraction, an extractionyield of matrine can be kept unchanged, but the acidic environment andthe heating condition during the extraction process will promote theacid hydrolysis of glycyrrhizic acid to form glycyrrhetinic acid, whichreduces an actual content of glycyrrhizic acid in a product by 18.7%.

B. Water is added, Scutellaria baicalensis roots are first added forextraction, and Sophora flavescens roots are then added for extraction;pH of a resulting solution is adjusted to 8 to 10 with 10% NaOH, andGlycyrrhiza inflata roots are added for extraction; a resulting solutionis filtered to obtain a filtrate and pH of the filtrate is adjusted to5.5 to 7.5; and then the filtrate is subjected to the treatments ofsteps S4 to S6. This solution will also lead to reduction in a contentof the active ingredient baicalin. As baicalin has extremely poor watersolubility, a strong alkaline condition is a must to achieve a highdissolution rate of baicalin. Furthermore, according to theaforementioned literature 1, a solubilizing effect of glycyrrhizic acidon baicalin cannot be exerted. Compared with the process of the presentinvention, an extraction yield of baicalin is reduced by 21.4%.

C. Water is added, Sophora flavescens roots are first added forextraction to obtain a Sophora flavescens root extraction solution, andthen 10% NaOH is added to adjust a pH to 9; Glycyrrhiza inflata rootsand Scutellaria baicalensis roots are added, a resulting mixture isheated to boiling, and extraction is conducted; a resulting solution isfiltered to obtain a filtrate and pH of the filtrate is adjusted to 5.5to 7.5; and then the filtrate is subjected to the treatments of steps S4to S6. This solution will also lead to a decrease of 36.1% in a contentof the active ingredient baicalin. On the surface, both glycyrrhizicacid and baicalin require alkaline conditions to be dissolved at a highrate, which seems plausible. In fact, the long-time stay in alkalineboiling water will promote the ring-opening variation of a C-ringstructure of baicalin, thereby greatly reducing an actual content ofbaicalin.

Therefore, if the three raw materials are added in a changed order,extraction yields of the main active ingredients in the medicinalmaterials will be significantly reduced, which will ultimately reducecontents of key active ingredients in a prepared composition and thusaffect the efficacy of the composition; and contents of inactiveingredients will be increased, which will lead to the increase of sideeffects.

Preferably, in step S4, the activated carbon may be added at an amount0.2% to 1% of the total amount of the filtrate; and the incubation maybe conducted at 85° C. to 95° C. for 0.5 h to 1 h.

In the above step, a resulting solution has a dark color, and activatedcarbon is used to remove part of pigments.

Preferably, in step S5, the clarifying agent may be one of chitosan, ZTCclarifying agent I-IV, and 101 juice clarifying agent, or a combinationof two or more thereof, the clarifying agent may be added at an amount1% to 5% of the total amount of the filtrate; and the standing may lastfor 0.5 h to 1 h.

In the above step, water is used as an extraction solvent. During waterextraction, in addition to small-molecule biologically activeingredients, a large number of water-soluble impurities in plants suchas sugars and proteins are also easily extracted, resulting in lowcontent of biologically active molecules. Moreover, sugars, proteins,etc. are prone to breed bacteria, and so a composition is easilydeteriorated after long-term storage. In the present invention, aclarifying agent is used to remove impurities prone to breed bacteria,so that a composition has a reduced risk of bacterium growth andbiologically active ingredients have increased contents and enhancedactivities.

The membrane separation may include the following: separating with amicrofiltration membrane; separating with an ultrafiltration membrane;and desalting with a reverse osmosis (RO) membrane.

Preferably, the method may further include the step of subjecting thecomposition obtained in step S6 to sterilization and/or preservativetreatment.

The sterilization may be one of high-temperature sterilization andmoist-heat sterilization. The composition is applicable to the field ofbiomedicines through different carriers.

A preservative used for the preservative treatment may be one of benzoicacid and benzoate, potassium sorbate, sodium dehydroacetate (SDHA), andsodium diacetate (SDA), or a combination of two or more thereof, or analcohol component with a preservative effect, which is applicable tofood, beverage, and health products; and the preservative may be addedat an amount 0.01% to 50% of the total amount of the filtrate.

More preferably, the alcohol component with a preservative effect mayinclude one of butanediol, 1,2-hexanediol, and glycerol, or acombination of two or more thereof, which is applicable to cosmetics.

The present invention also provides use of a composition prepared by themethod described above in foods, drugs, and cosmetics.

The composition prepared in the present invention, when used incosmetics, can enhance the inhibition of inflammation, thus relievingskin sensitivity; can also repair cell damage so as to achieve theeffect of improving skin cell vitality; can also promote the cell geneexpression to repair a skin barrier, thus reducing skin diseases causedby external stimuli.

Compared with the prior art, the present invention has the followingbeneficial effects.

1) In the present invention, the three medicinal materials (Sophoraflavescens roots, Glycyrrhiza inflata roots, and Scutellaria baicalensisroots) are fed in sequence for co-extraction, so that the activeingredients of the three medicinal materials promote each other'sextraction efficiency and stability during the whole extraction process.Compared with the method of subjecting each of the medicinal materialsto extraction alone and the method of subjecting any two of themedicinal materials to extraction, the present invention requires lessprocess steps, reduced energy consumption, low cost, and simpleoperations, and thus is suitable for continuous and large-scaleproduction. The method of the present invention is anenvironmentally-friendly, promising extraction technology forbiologically active ingredients, and is suitable for industrializedproduction.

2) In the present invention, the three medicinal materials (Sophoraflavescens roots, Glycyrrhiza inflata roots, and Scutellaria baicalensisroots) are fed in sequence for co-extraction, which improves theextraction efficiency of active ingredients and shortens a process.Therefore, a process can be completed in a short time to reduce the riskthat water-soluble samples are contaminated by bacteria due to long-timeproduction, which is conducive to the use and preservation of products.

3) The present invention does not use organic solvents in the process ofextraction, separation, and purification, so that the solvent residuesin medicines, health products, foods, and cosmetics are reduced, whichis beneficial to the widespread use of products; and the recovery,storage, and discharge of organic solvents are reduced, which isbeneficial to the use of factory production equipment and facilitatesenvironmental protection and energy conservation.

4) In addition to the necessary washing wastewater generated during thepreparation process, all solvents used in the process serve as solventsfor products, and no other wastewater is generated, which is energysaving and environmentally friendly.

5) Through the above-mentioned method where the three medicinalmaterials (Sophora flavescens roots, Glycyrrhiza inflata roots, andScutellaria baicalensis roots) are fed in sequence for co-extraction inthe present invention, contents of biologically active ingredients areincreased, and the ingredients are more stable in a solution due tomutual promotion of molecules, making a final product have improved andexpanded efficacy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing inhibition rates of different test substancesamples at a concentration of 0.3125%; and

FIG. 2 is a diagram showing inhibition rates of different test substancesamples at a concentration of 0.078%.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present invention is described in detail below with reference tospecific examples. The following examples will help those skilled in theart to further understand the present invention, but do not limit thepresent invention in any way. It should be noted that those of ordinaryskill in the art can further make several variations and improvementswithout departing from the idea of the present invention. These all fallwithin the protection scope of the present invention.

Example 1

An environmentally-friendly preparation method of a compositioncontaining anti-inflammatory active ingredients included the followingsteps:

(1) 1,000 g of water was weighed and added to an extracting tank, a pHwas adjusted to 4.5 with dilute HCl, and a resulting solution was heatedto 80° C.; and then 40 g of Sophora flavescens roots was weighed andadded to the extracting tank, and extraction was conducted for 2 h at80° C.;

(2) 10% NaOH was added to a solution obtained in step (1) to adjust a pHto 10, and then 40 g of Glycyrrhiza inflata roots was added; and aresulting mixture was heated to boiling, and extraction was conductedfor 30 min;

(3) 40 g of Scutellaria baicalensis roots was added to a solutionobtained in step (2), and extraction was conducted for 2 h; and aresulting solution was filtered to obtain a filtrate, and a pH of thefiltrate was adjusted to 7.5;

(4) 1% activated carbon was added to the filtrate obtained in step (3),and a resulting mixture was heated to 95° C.; reaction was conducted atthe temperature for 1 h; and a resulting reaction solution was filteredto obtain a filtrate;

(5) a 5% ZTC clarifying agent II was added to the filtrate obtained instep (4), and a resulting mixture stood for 1 h and filtered;

(6) a filtrate obtained in step (5) was filtered through amicrofiltration membrane to remove suspended solids, bacteria, etc.,then through an ultrafiltration membrane to remove macromolecularorganics, proteins, etc., and finally through an RO membrane fordesalination to obtain a filtrate;

(7) butanediol was added to the filtrate obtained in step (6) to obtaina light-yellow transparent liquid with a butanediol content of 35%,namely, the composition.

In the obtained composition, matrine has an extraction yield of 95%,glycyrrhizic acid in Glycyrrhiza inflata has an extraction yield of 94%,and baicalin has an extraction yield of 91%.

Example 2

An environmentally-friendly preparation method of a compositioncontaining anti-inflammatory active ingredients included the followingsteps:

(1) 1,000 g of water was weighed and added to an extracting tank, a pHwas adjusted to 4 with dilute HCl, and a resulting solution was heatedto 70° C.; and then 30 g of Sophora flavescens roots was weighed andadded to the extracting tank, and extraction was conducted for 1 h at70° C.;

(2) 10% NaOH was added to a solution obtained in step (1) to adjust a pHto 9, and then 40 g of Glycyrrhiza inflata roots was added; and aresulting mixture was heated to boiling, and extraction was conductedfor 60 min;

(3) 30 g of Scutellaria baicalensis roots was added to a solutionobtained in step (2), and extraction was conducted for 1 h; and aresulting solution was filtered to obtain a filtrate, and a pH of thefiltrate was adjusted to 6.5;

(4) 0.5% activated carbon was added to the filtrate obtained in step(3), and a resulting mixture was heated to 90° C.; reaction wasconducted at the temperature for 45 min; and a resulting reactionsolution was filtered to obtain a filtrate;

(5) a 0.5% chitosan clarifying agent was added to the filtrate obtainedin step (4), and a resulting mixture stood for 45 min and filtered;

(6) a filtrate obtained in step (5) was filtered through amicrofiltration membrane to remove suspended solids, bacteria, etc.,then through an ultrafiltration membrane to remove macromolecularorganics, proteins, etc., and finally through an RO membrane fordesalination to obtain a filtrate;

(7) butanediol was added to the filtrate obtained in step (6) to obtaina light-yellow transparent liquid with a butanediol content of 35%,namely, the composition.

In the obtained composition, matrine has an extraction yield of 96%,glycyrrhizic acid in Glycyrrhiza inflata has an extraction yield of 96%,and baicalin has an extraction yield of 93%.

Example 3

An environmentally-friendly preparation method of a compositioncontaining anti-inflammatory active ingredients included the followingsteps:

(1) 1,000 g of water was weighed and added to an extracting tank, a pHwas adjusted to 3 with dilute HCl, and a resulting solution was heatedto 60° C.; and then 21 g of Sophora flavescens roots was weighed andadded to the extracting tank, and extraction was conducted for 30 min at60° C.;

(2) 1% NaOH was added to a solution obtained in step (1) to adjust a pHto 8, and then 21 g of Glycyrrhiza inflata roots was added; and aresulting mixture was heated to boiling, and extraction was conductedfor 120 min;

(3) 21 g of Scutellaria baicalensis roots was added to a solutionobtained in step (2), and extraction was conducted for 30 min; and aresulting solution was filtered to obtain a filtrate, and a pH of thefiltrate was adjusted to 5.5;

(4) 0.2% activated carbon was added to the filtrate obtained in step(3), and a resulting mixture was heated to 85° C.; reaction wasconducted at the temperature for 0.5 h; and a resulting reactionsolution was filtered to obtain a filtrate;

(5) a 1% ZTC clarifying agent II was added to the filtrate obtained instep (4), and a resulting mixture stood for 0.5 h and filtered;

(6) a filtrate obtained in step (5) was filtered through amicrofiltration membrane to remove suspended solids, bacteria, etc.,then through an ultrafiltration membrane to remove macromolecularorganics, proteins, etc., and finally through an RO membrane fordesalination to obtain a filtrate;

(7) butanediol was added to the filtrate obtained in step (6) to obtaina light-yellow transparent liquid with a butanediol content of 35%,namely, the composition.

In the obtained composition, matrine has an extraction yield of 96%,glycyrrhizic acid in Glycyrrhiza inflata has an extraction yield of 97%,and baicalin has an extraction yield of 94%.

Comparative Example 1

30 g of Sophora flavescens roots and 40 g of Glycyrrhiza inflata rootswere simultaneously fed, and then extraction was conducted with water at80° C. for 2 h to obtain a filtrate 1. 30 g of Scutellaria baicalensisroots alone was subjected to extraction at 80° C. for 2 h to obtain afiltrate 2. The filtrate 1 and the filtrate 2 were mixed, and aresulting mixture was subjected to the treatments of steps (4) to (7) inExample 1 to obtain a composition.

Comparative Example 2

30 g of Scutellaria baicalensis roots and 40 g of Glycyrrhiza inflataroots were simultaneously fed and then extraction was conducted withwater at 80° C. for 2 h to obtain a filtrate 1. 30 g of Sophoraflavescens roots alone was subjected to extraction at 80° C. for 2 h toobtain a filtrate 2. The filtrate 1 and the filtrate 2 were mixed, and aresulting mixture was subjected to the treatments of steps (4) to (7) inExample 1 to obtain a composition.

Comparative Example 3

30 g of Scutellaria baicalensis roots and 30 g of Sophora flavescensroots were simultaneously fed and then extraction was conducted withwater at 80° C. for 2 h to obtain a filtrate 1. 30 g of Glycyrrhizainflata roots alone was subjected to extraction at 80° C. for 2 h toobtain a filtrate 2. The filtrate 1 and the filtrate 2 were mixed, and aresulting mixture was subjected to the treatments of steps (4) to (7) inExample 1 to obtain a composition.

Effect Verification:

The composition solutions obtained in Example 1 and Comparative Examples1, 2, and 3 were tested for efficacy on skin-related cells. Specificresults were as follows.

1. Cytotoxicity

Cytotoxicity refers to a deleterious effect on cell structure and/orbasic processes of cell survival, proliferation and function. Toxicityis a result of non-specific changes in basic cell functions (such asmitochondria, lysosomes, and plasma membrane integrity), which mayeventually lead to changes in organ-specific functions or death of anorganism.

The mouse macrophage cell line Raw 264.7 has strong ability to adhereand swallow antigens, which is a cell line commonly used for the studyof microbiology and immunology and is also one of the most classic celllines for the study of anti-inflammatory effects. Neutral red (NR) is aweakly-cationic in vitro reactive dye, which easily diffuses through theplasma membrane and accumulates in the lysosome, and combines with theanionic lysosome matrix to form an electronically-stable state. Toxicsubstances change a cell surface or lysosomal membrane, causinglysosomal fragility and other harmful changes that are graduallyirreversible. These harmful changes can cause cell death and/or inhibitcell growth, thereby leading to a decrease in the amount of NR stored incells. Therefore, NR uptake experiments can reflect cytotoxicity.

In this experiment, the toxicity of the compositions obtained in theexamples and comparative examples to Raw 264.7 cells was first studiedto evaluate the safety of the compositions. Main reagents andinstruments used in the experiment were shown in Table 1. Raw264.7 cellswere used for the NR uptake experiment.

TABLE 1 Main reagents and instruments used in the experiment Name ofinstruments and reagents Brand Model Raw264.7 cell ATCC TIB-71 DMEMmedium Gibco 10569-010 Fetal bovine serum (FBS) Corning 35-076-CVPenicillin/streptomycin (PS) Corning 15140-122 0.25% trypsin Gibco25200-056 Phosphate-buffered saline (PBS) Sigma P4417-50TAB NR dye SigmaN7005-1G Inverted microscope Jiangnan Yongxing XD202 Cell counter ThermoCountess II 96-well plate Corning 3599 Carbon dioxide incubator Thermo 371 Microplate shaker Kylin-bell QB9001 Microplate reader ThermoMultiSkan FC

Raw264.7 cells at logarithmic growth phase were collected as follows:culture medium was discarded, and the cells were washed twice with PBS,added with 0.25% trypsin, and placed at 37° C. for 5 min; digestion wasterminated with DMEM complete culture medium including 10% FBS and 1%PS, and a resulting solution was centrifuged at 1,000 rpm for 3 mi; anda resulting supernatant was discarded, and obtained cells wereresuspended in complete culture medium and counted. A cell concentrationwas adjusted to 5×10⁵ cells/ml, and a resulting cell suspension wasadded to a 96-well plate at 100 μl/well. After the cells grew adherently24 h later, 100 μl of each of test substance samples with differentconcentrations was added to each well. A resulting mixture wasthoroughly mixed and incubated for 24±1 h in an incubator.

Experimental groups were shown in Table 2 below:

TABLE 2 Group name Sample treatment Blank control group Complete culturemedium Negative control group Complete culture medium + Raw264.7 cellsTest substance group Complete culture medium + Raw264.7 cells + testsubstance Notes: The test substances were composition solutions ofdifferent concentrations obtained by diluting the compositions preparedin Examples 1 to 3 and Comparative Examples 1 to 3 (starting from aconcentration of 5%, 4-fold dilution was adopted to obtain finalconcentrations: 5%, 1.25%, 0.3125%, and 0.078%).

After the treatment, the culture medium was discarded, 200 μl of an NRstaining solution (33 μg/ml) was added to each well, and the plate wasplaced in an incubator for 3±0.1 h. A resulting supernatant wasdiscarded (no residue), 150 μl of an NR eluent (1% glacial aceticacid+50% absolute ethanol+4900 ultrapure water) was added to each well,and the plate was shaken at room temperature for 20 min to 45 min in thedark. An absorbance (A) was determined at 540 nm with a microplatereader, and a cell viability was calculated. Cell viability(0%)=A_(test substance group)/A_(negative control)×100%

As shown in Table 3, after Raw 264.7 cells were treated for 24 h withcomposition solutions at a concentration of 1.25% obtained by dilutingthe compositions prepared in examples and comparative examples, exceptfor Comparative Example 1, the cell viability in the remaining groupsdecreased to 60% or lower, indicating that the compositions exhibited aspecified toxic effect on cells at a high concentration. After the cellswere treated with each composition at a concentration of 10.3125, thecell activity in each example was significantly higher than that in the3 comparative examples, indicating that the cytotoxicity of the exampleswas relatively low.

TABLE 3 Effects of compositions at different concentrations on cellviability Cell Cell Sample Concentration viability Concentrationviability treatment 1 (%) 1 (%) 2 (%) 2 (%) Negative control — 100.00 —100.00 group Comparative 1.25 104.92 0.3125 100.73 Example 1 Comparative1.25 21.30 0.3125 32.48 Example 2 Comparative 1.25 20.78 0.3125 31.10Example 3 Example 1 1.25 13.69 0.3125 116.20 Example 2 1.25 32.59 0.3175108.44 Example 3 1.25 55.34 0.3175 106.31

2. Anti-Inflammatory Effect

Inflammation refers to a basic pathological process (mainly defensiveresponse) of a living tissue with a vascular system to the stimulationof various damage factors. The clinical manifestations of inflammationinclude a series of phenomena such as redness, swelling, fever, itching,and pain.

Lipopolysaccharide (LPS) is one of the main components in the cell wallof Gram-negative bacilli and is also one of the main substances thatinduce inflammation. In an inflammatory response process, macrophagesindirectly or directly participate in response processes of variousinflammatory diseases by generating a variety of different cytokines orreleasing lysosomal enzymes, and can respond to the stimulation ofextracellular LPS and initiate a series of cascade reactions in cellscaused by the activation of corresponding signal proteins to producevarious inflammatory mediators, such as prostaglandin E2 (PGE-2) andnitric oxide (NO).

In this experiment, Raw 264.7 cells were stimulated by LPS, then cellculture was collected, and the expression of the inflammatory factorPGE-2 was determined by enzyme-linked immunosorbent assay (ELISA). Thecompositions obtained in the examples and comparative examples werecompared in terms of the influence on anti-inflammatory effects. Mainreagents and instruments used in the experiment were shown in Table 4.Raw264.7 cells and LPS were used for the experiment.

TABLE 4 Main reagents and instruments used in the experiment. Name ofinstruments and reagents Brand Model Raw264.7 cell ATCC TIB-71 DMEMmedium Gibco 10569-010 FBS Corning 35-076-CV PS Corning 15140-122 0.25%trypsin Gibco 25200-056 PBS Sigma P4417-50TAB LPS Sigma L3012-10MGDexamethasone (Dex) Sigma D4902-25MG PGE-2 ELISA kit Cayman 514010 Inverted microscope Jiangnan Yongxing XD202 Cell counter Thermo CountessII 96-well plate Corning 3599 Carbon dioxide incubator Thermo  371Microplate shaker Kylin-bell QB9001 Microplate reader Thermo MultiSkanFC

Raw264.7 cells at logarithmic growth phase were collected as follows:culture medium was discarded, and the cells were washed twice with PBS,added with 0.25% trypsin, and placed at 37° C. for 5 min; digestion wasterminated with DMEM complete culture medium including 10% FBS and 1%PS, and a resulting solution was centrifuged at 1,000 rpm for 3 min; anda resulting supernatant was discarded, and obtained cells wereresuspended in complete culture medium and counted. A cell concentrationwas adjusted to 5×10⁵ cells/ml, and a resulting cell suspension wasadded to a 96-well plate at 100 l/well. After the cells grew adherently24 h later, 100 μl of each of test substance samples with differentconcentrations, positive control, negative control, and 1 μg/ml LPS wereadded to each well. A resulting mixture was thoroughly mixed andincubated for 24 h±1 h in an incubator. According to cytotoxicityresults, a concentration (less than 0.3125%) that shows little effect oncell activity was selected for the verification of the anti-inflammatoryexperiment. Experimental groups were shown in Table 5 below:

TABLE 5 Group name Sample treatment Blank control group Complete culturemedium Negative control group Complete culture medium + Raw264.7 cellsLPS group (model group) Complete culture medium + Raw264.7 cells + LPSPositive control group Complete culture medium + Raw264.7 cells + LPS +Dex Test substance group Complete culture medium + Raw264.7 cells +LPS + test substance Notes: The test substances were compositionsolutions of different concentrations (0.3125% and 0.078%) obtained bydiluting the compositions prepared in Example 1 and Comparative Examples1 to 3. Dex refers to dexamethasone (10 μM).

After the treatment, cell culture was collected to a new 96-well plate,and the expression of PGE-2 was determined by ELISA according toinstructions of a kit. A PGE-2 content (E) was calculated according to astandard curve, and relative to the LPS group, a relative inhibitionrate was calculated for each sample (inhibition rate(%)=(1-E_(test substance group)/E_(LPS group))×100).

As shown in FIG. 1 and FIG. 2, on Raw 264.7 cells, LPS significantlyinduces the expression of PGE-2. The positive control Dex can inhibitthe expression of PGE-2 in a dose-dependent manner, and can achieve aninhibition rate reaching 75.27% at 10 μM (Dex-10 group shown in FIG. 1).A liquid plant extract composition prepared in Example 1 can reduce theexpression of PGE-2 in a dose-dependent manner in a range of 0.078% to0.3125%, exhibiting a statistically significant difference from the 3comparative examples (** P<0.01). The composition can achieve aninhibition rate of 72.00% at a concentration of 0.3125% and aninhibition rate of 41.17% at a concentration of 0.078%, which isequivalent to the anti-inflammatory effect of the positive control Dex.

In summary, compared with Comparative Examples 1 to 3, the compositionprepared in Example 1 shows a lower toxicity to mouse macrophage cellsRaw 264.7 and a stronger inhibitory effect on the expression of theinflammatory factor PGE-2 induced by LPS.

As determined, the compositions prepared in Examples 2 and 3 also show alow toxicity to mouse macrophage cells Raw 264.7 and a strong inhibitoryeffect on the expression of the inflammatory factor PGE-2 induced byLPS.

There are many ways to specifically apply the present invention, and theabove are merely preferred implementations of the present invention. Itshould be noted that the foregoing examples are provided only forillustrating the present invention and are not intended to limit theprotection scope of the present invention. For a person of ordinaryskill in the art, several improvements may further be made withoutdeparting from the principle of the present invention, and theseimprovements should also be considered as falling within the protectionscope of the present invention.

What is claimed is:
 1. An environmentally-friendly preparation method ofa composition containing anti-inflammatory active ingredients,comprising the following steps: S1. weighing a predetermined volume ofwater and adjusting a pH of the predetermined volume of water, addingSophora flavescens roots to obtain a first resulting mixture, andsubjecting the first resulting mixture to a first extraction at apredetermined temperature to obtain an extracted solution; S2. adjustinga pH of the extracted solution obtained in step S1, adding Glycyrrhizainflata roots to obtain a second resulting mixture, and heating thesecond resulting mixture to boiling for a second extraction to obtain anextracted boiling solution; S3. adding Scutellaria baicalensis roots tothe extracted boiling solution obtained in step S2 to obtain a thirdresulting mixture, and subjecting the third resulting mixture to a thirdextraction, a first filtration, and a pH adjustment to obtain a firstfiltrate; S4. adding activated carbon to the first filtrate obtained instep S3 to obtain a fourth resulting mixture, and subjecting the fourthresulting mixture to an incubation and a second filtration to obtain asecond filtrate; S5. adding a clarifying agent to the second filtrateobtained in step S4 to obtain a fifth resulting mixture, and subjectingthe fifth resulting mixture to a standing and a third filtration toobtain a third filtrate; and S6. subjecting the third filtrate obtainedin step S5 to a membrane separation to obtain the composition containingthe anti-inflammatory active ingredients.
 2. Theenvironmentally-friendly preparation method according to claim 1,wherein, the Scutellaria baicalensis roots, the Sophora flavescens rootsand the Glycyrrhiza inflata roots are added at a ratio of(21-40):(21-40):(21-40).
 3. The environmentally-friendly preparationmethod according to claim 1, wherein, in step S1, the predeterminedvolume of water is added at an amount 5 to 10 times a total amount ofthe Scutellaria baicalensis roots, the Sophora flavescens roots and theGlycyrrhiza inflata roots; the pH of the predetermined volume of wateris 3.0 to 7.0; and the first extraction is conducted at thepredetermined temperature of 60° C. to 80° C. for 30 min to 2 h.
 4. Theenvironmentally-friendly preparation method according to claim 1,wherein, in step S2, the pH of the extracted solution is adjusted to 8to 10 with 1% to 10% NaOH, and the second resulting mixture is subjectedto the second extraction for 30 min to 2 h.
 5. Theenvironmentally-friendly preparation method according to claim 1,wherein, in step S3, the third extraction is conducted for 30 min to 2h, and a pH of the third resulting mixture is adjusted to 5.5 to 7.5. 6.The environmentally-friendly preparation method according to claim 1,wherein, in step S4, the activated carbon is added at an amount 0.2% to1% of a total amount of the first filtrate; and the incubation isconducted at 85° C. to 95° C. for 0.5 h to 1 h.
 7. Theenvironmentally-friendly preparation method according to claim 1,wherein, in step S5, the clarifying agent is at least one selected fromthe group consisting of chitosan, ZTC clarifying agent I-IV, and 101juice clarifying agent; the clarifying agent is added at an amount 1% to5% of a total amount of the second filtrate; and the standing lasts for0.5 h to 1 h.
 8. The environmentally-friendly preparation methodaccording to claim 1, wherein, in step S6, the membrane separationcomprises: separating with a microfiltration membrane; separating andconcentrating with an ultrafiltration membrane; and desalting with areverse osmosis (RO) membrane.
 9. The environmentally-friendlypreparation method according to claim 1, wherein, theenvironmentally-friendly preparation method further comprises the stepof subjecting the composition containing the anti-inflammatory activeingredients obtained in step S6 to a sterilization and/or a preservativetreatment; the sterilization is one of a high-temperature sterilizationand a moist-heat sterilization; and a preservative used for thepreservative treatment is at least one selected from the groupconsisting of benzoic acid and benzoate, potassium sorbate, sodiumdehydroacetate (SDHA), and sodium diacetate (SDA), or the preservativeis an alcohol component with a preservative effect; and the preservativeis added at an amount 0.01% to 50% of a total amount of the thirdfiltrate.
 10. A method of using the composition containing theanti-inflammatory active ingredients prepared by theenvironmentally-friendly preparation method according to claim 1,comprising using the composition containing the anti-inflammatory activeingredients in health foods, drugs, and cosmetics.